Nucleic acid amplification techniques have opened broad new approaches to genetic testing and DNA analysis, e.g. Arnheim and Erlich, Ann. Rev. Biochem., 61: 131-156 (1992). PCR in particular has become a research tool of major importance with applications in cloning, analysis of genetic expression, DNA sequencing, genetic mapping, drug discovery, and the like, e.g. Arnheim et al (cited above); Gilliland et at, Proc. Natl. Acad. Sci., 87:2725-2729 (1990); Bevan et at, PCR Methods and Applications, 1:222-228 (1992); Green et at, PCR Methods and Applications, 1: 77-90(1991); Blackwell et al, Science, 250:1104-1110 (1990). The widespread applications of such nucleic acid amplification techniques has driven the development of instrumentation for carrying out the amplification reactions under a variety of circumstances. Important design goals for such instrument development have included fine temperature control, minimization of sample-to-sample variability in multi-sample thermal cycling, automation of pre- and post-reaction processing steps, high speed temperature cycling, minimization of sample volumes, real time measurement of amplification products, minimization of cross-contamination, or "sample carryover," and the like. In particular, the design of instruments permitting amplification to be carried out in closed reaction chambers and monitored in real time would be highly desirable for preventing cross-contamination, e.g. Higuchi et al, Biotechnology, 10:413-417 (1992) and 11: 1026-1030(1993); and Holland et al, Proc. Natl. Acad. Sci., 88: 7276-7280 (1991). Clearly, the successful realization of such a design goal would be especially desirable in the analysis of diagnostic samples, where a high frequency of false positives and false negatives--caused by "sample carryover"--would severely reduce the value of an amplification procedure. Moreover, real time monitoring of an amplification reaction permits far more accurate quantitation of starting target DNA concentrations in multiple-target amplifications, as the relative values of close concentrations can be resolved by taking into account the history of the relative concentration values during the reaction. Real time monitoring also permits the efficiency of the amplification reaction to be evaluated, which can indicate whether reaction inhibitors are present in a sample.
Holland et al (cited above) and others have proposed fluorescence-based approaches to provide real time measurements of amplification products during a PCR. Such approaches have either employed intercalating dyes (such as ethidium bromide) to indicate the amount of double stranded DNA present, or they have employed probes containing fluorester-quencher pairs (the so-called "Tac-Man" approach) that are cleaved during amplification to release a fluorescent product whose concentration is proportional to the amount of double stranded DNA present.
The latter approach, illustrated in FIG. 1, involves the use of an oligonucleotide probe that specifically anneals to a region of the target polynucleotide "downstream," i.e. in the direction of extension, of primer binding sites. The probe contains a fluorescent "reporter" molecule and a "quencher" molecule such that the whenever the reporter molecule is excited, the energy of the excited state nonradiatively transfers to the quencher molecule where it either dissipates nonradiatively or is emitted at a different emission frequency than that of the reporter molecule. During strand extension by a DNA polymerase, the probe anneals to the template where it is digested by the 5'-&gt;3' exonuclease activity of the polymerase. Upon digestion, the quencher molecule is no longer close enough to the reporter molecule to quench emissions by energy transfer. Thus, as more and more probe gets digested during amplification, a stronger and stronger fluorescent signal is generated.
Three main factors determine the performance of such a doubly labeled fluorescent probe: First is the degree of quenching observed in the intact unbound probe. This can be characterized by the ratio, designated herein as "RQ.sup.- ", of fluorescent emissions of the reporter molecule and the quencher molecule absent hybridization to a complementary polynucleotide. That is, RQ.sup.- is the ratio of fluorescent emissions of the reporter molecule and the quencher molecule when the S oligonucleotide probe is in a single stranded state. Influences on the value of RQ.sup.- include the particular reporter and quencher molecules used, the spacing between the reporter and quencher molecules, nucleotide sequence-specific effects, the degree of flexibility of structures, e.g. linkers, to which the reporter and quencher molecules are attached, the presence of impurities, and the like, e.g. Wu et at, Anal. Biochem., 218: 1-13 (1994); and Clegg, Meth. Enzymol., 211:353-388 (1992). (A related quantity, RQ.sup.+, is the ratio of fluorescent emissions of the reporter molecule and the quencher molecule when the oligonucleotide probe is in a double stranded state with a complementary polynucleotide). A second factor is the efficiency of hybridization, which depends on probe melting temperature, T.sub.m, the presence of secondary structure in the probe or target polynucleotide, annealing temperature, and other reaction conditions. Finally, a third factor is the efficiency at which the DNA polymerase 5'.fwdarw.3' exonuclease activity cleaves the bound probe between the reporter molecule and quencher molecule. Such efficiency depends on the proximity of the reporter or quencher to the 5' end of the probe, the "bulkiness" of the reporter or quencher, the degree of complementarity between the probe and target polynucleotide, and like factors, e.g. Lee et al, Nucleic Acids Research, 21:3761-3766 (1993).
As quenching is completely dependent on the physical proximity of the reporter molecule and quencher molecule, it has been assumed that the quencher and reporter molecules must be attached to the probe within a few nucleotides of one another, usually with a separation of about 6-16 nucleotides, e.g. Lee et al (cited above); Mergny et at, Nucleic Acids Research, 22:920-928 (1994); Cardullo et at, Proc. Natl. Acad. Sci., 85:8790-8794 (1988); Clegg et at, Proc. Natl. Acad. Sci., 90:2994-2998 (1993); Ozaki et at, Nucleic Acids Research, 20:5205-5214 (1992); and the like. Typically, this separation is achieved by attaching one member of a reporter-quencher pair to the 5' end of the probe and the other member to a base 6-16 nucleotides away. Unfortunately, there are at least two significant drawbacks to this arrangement. First, attaching reporter or quencher molecules typically involves more difficult chemistry than, for example, that used to attach moieties to an end. And second, attachment of reporter or quencher molecules to internal nucleotides adversely affects hybridization efficiency, e.g. Ward et at, U.S. Pat. No. 5,328,824; Ozaki et al (cited above); and the like.
In view of the above, the application of techniques for real-time monitoring of nucleic acid amplification would be facilitated by the availability of a conveniently synthesized probe having efficient hybridization characteristics and distinct fluorescent characteristic in a bound double stranded state and an unbound single stranded state.